Chemokine receptors are expressed on the surface of certain cells, which interact with cytokines called chemokines. The CXC chemokine receptor 4 (CXCR4) is a G-protein-coupled receptor that transduces signals of its endogenous ligand, the chemokine CXCL12 (stromal cell-derived factor-1, SDF-I). Following interaction of CXCR4/CXCL12, intracellular calcium (Ca2+) ions fluxes are triggered. This causes cellular responses, including chemotaxis, allowing cells to travel within the organism.
CXCR4 is expressed on myeloid cells, T-lymphocytes, B-lymphocytes, epithelial cells, endothelial cells and dendritic cells. Thus, the expression of this molecule on the surface of tumor cells make it a suitable candidate as ligand for the specific targeting of therapeutic compounds to cells expressing said molecule. For instance, WO2006029078 describes fusion constructs comprising a protein translocation domain formed by the Tat protein, the CXCR4-receptor binding DV3 peptide domain and a therapeutic agent which is either a cdk2 antagonist peptide or a p53 activating peptide. These constructs are targeted to cells expressing CXCR4 and, by means of the protein translocation domain, the therapeutic agent is translocated inside the cell. However, these constructs require, in addition to the CXCR4 ligand which acts solely in the docking of the construct to the cell, a translocating domain which delivers the therapeutic agent inside the cell.
Driessen et al. (Molecular Therapy, 2008, 16:516-524) describes the use of a peptide analog, 4-fluorobenzoyl-RR-(L-3-(2-naphthyl) alanine)-CYEK-(1-citrulline)-PYR-(1-citrulline)-CR (SEQ ID NO: 1), covalently linked to a phospholipid to target a lipid-based gene delivery vehicle to CXCR4+-cells. However, this method shows low efficiency and requires increasing expression of CXCR4 on the surface of the target cells by contacting the cells with VEGF prior to the contacting with the conjugates.
Le Bon et al. (Bioconjugate Chem. 2004, 15, 413-423) describe the use of the CXCR4 specific ligands AMD3100 and AMD3100 for promoting specific gene transfer into cells expressing CXCR4 using lipid and polycationic conjugates. However, this method requires the conjugation of a phorbol ester derivative to the polycationic lipid therapeutic agent in order to increase CXCR4 expression on the surface of the target cells.
Egorova et al. (J Gene Med 2009; 11: 772-781) describe conjugates formed by CXCR4 ligands (the peptides KPVSLSYRSPSRFFESH-K9-biotin [(SEQ ID NO: 2)-biotin], KPVSLSYR-K9-biotin [(SEQ ID NO: 3)-biotin] and D-LGASWHRPDK-K9-biotin [(SEQ ID NO: 4)-biotin] and DNA that binds the polylysine region electrostatically and the use thereof for delivery of nucleic acids to CXCR4 positive cells. However, these conjugates have low efficiency and rely on the use of peptides showing agonistic activity towards CXCR4 which may result in an increased tumor proliferation as a response of the stimulation of CXCR4 by the ligands.
Tamamura et al. (Bioinorganic & Medicinal Chemistry, 2001, 9: 2179-2187) describe conjugates of different analoges of the T140 CXCR4 agonist and 3′-azido-3-deoxythymidine (AZT) and their use for preventing HIV-1-induced cytopathogenicity in MT-4 cells. However, no evidence was provided that these conjugates were capable of targeting CXCR4-expressing cells in vivo or that the AZT conjugated to the CXCR4 ligand can be internalized by CXCR4.
Moreover, all these conjugates act by delivering the therapeutic agent to the surface of the cell from where internalization of the said agent requires its binding to specific receptors on the cell surface. Therefore, there is a need in the art for further conjugates suitable for the specific delivery of molecules of interest to CXCR4 cells which overcome the problems of the conjugates described in the prior art and wherein internalization of the therapeutic agent occurs by the use of a CXCR4-targeting molecule which can be internalized by the CXCR4-expressing cells together with the therapeutic agent bound to it.